Methods and Compounds for Promoting Vessel Regression

ABSTRACT

The present invention relates, at least in part, to methods and compositions for treating and diagnosing disorders associated with neovascularization, and methods for identifying targets and compositions used in treating and diagnosing such disorders.

CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.11/363,402, which was filed on Feb. 24, 2006, which claims the benefitunder 35 USC §119(e) of U.S. Provisional Patent Application Ser. Nos.60/656,168, filed on Feb. 24, 2005, and 60/719,998, filed on Sep. 23,2005. The entire contents of each of these applications is herebyincorporated by reference.

TECHNICAL FIELD

This invention relates to methods and compounds for promoting vesselregression, e.g., to treat disorders associated with neovascularization.

BACKGROUND

The hyaloid vascular system (HVS) is a transiently existing network ofcapillaries that function to nourish the immature lens and primaryvitreous of the developing eye. The hyaloid artery (HA) runs from theback of the eye to the embryonic lens giving rise to a capillary plexusthat surrounds the lens, consisting of the vasa hyaloidea propria (VHP),the tunica vasculosa lentis (TVL) and the pupillary membrane (PM). TheHVS provides a useful system to investigate physiologically relevantangiogenesis and vascular remodeling processes.

The first elements of the human hyaloid vasculature to undergoregression are the VHP, followed by the TVL, PM and lastly the mainhyaloid trunk, commencing at 12 weeks of gestation (WG) and culminatingin the involution of the entire hyaloid by 35-36 WG (Mann I. TheDevelopment of the Human Eye. Pp 201-32. Grune & Stratton Inc: New York,1964.). In humans, failure of the hyaloid vascular system to regress canlead to a condition known as persistent hyperplastic primary vitreous(PHPV), which can result in permanent blindness if left untreated.

Mitchell and coworkers showed that in mice the hyaloid vascular systemis first recognized at embryonic day 10.5 (E10.5), is complete by E13.5and regresses postnatally prior to eyelid opening (Mitchell C A, et al.,Dev. Dyn. 213(3):322-33 (1998)). During mouse development, the longerbranches of the TVL and the longer hyaloid vessels are removed bypost-gestational day 16 (P 16) (Mitchell C A, et al., Dev. Dyn.213(3):322-33 (1998)). Ito et al. presented partially similar results,showing that the PM had completely disappeared by P16, and the VHP haddisappeared between P12 and P16, but the TVL and the hyaloid arteryremained even at P16 (Ito and Yoshioka, Anat. Embryol. (Berl).200(4):403-11 (1999)). Smith showed a gradual disappearance of the TVLand hyaloid artery from P14 to P30 (Smith, Systematic evaluation of theMouse Eye: Anatomy, Pathology, and Biomethods. Pp 45-63. Sunders,2002.).

Previous hypotheses of the mechanism of regression of the hyaloidvascular system included the following:

A. The reduction or the cessation of the blood flow into the HVS wasthought to be one of the major triggering factors of the regression inthese vessels. A change in the blood flow distribution (Bischoff et al.,Graefes Arch. Clin. Exp. Ophthalmol. 220 (6):257-63 (1983)) andcompetition between the blood vessels in the retina and the lens for theblood flow can lead to the degeneration of the HVS when the retinalblood vessels become larger and require more nutrients.

B. Vascular obstruction, physical vascular stretching, localizedcirculatory stasis, and arterial vasoconstriction were regarded astriggering factors of the regression of the HVS (Jack, Am. J.Ophthalmol. 74 (2):261-72 (1972); Latker and Kuwabara, Invest.Ophthalmol. Vis. Sci. 21 (5):689-99 (1981)). The regression occurs firstin those vessels that were hemodynamically disadvantaged andconsequently had less blood flow.

C. Meeson et al. have shown that the occurrence of apoptosis in the PMstrictly correlated to the flow status; as the flow decreased theappearance of apoptosis in capillaries increased (Meeson et al.,Development. 122(12):3929-38 (1996)). The same correlation may exist inthe hyaloid vascular system. As the main role of the HVS is supposed tobe to nourish the retina before the maturation of retinal vessels, theHVS may regress after the completion of these vessels.

D. Macrophages may also be required in the programmed regression of thePM and the HVS (Lang and Bishop, Cell. 13; 74 (3):453-62 (1993); Lang etal., Development. 120 (12):3395-403 (1994); Diez-Roux and Lang,Development. 124 (18):3633-8 (1997)).

E. The anti-angiogenic ability of the vitreous humor and vitreousextracts may also be important in the regression of the TVL (Preis etal., Am. J. Ophthalmol. 84 (3):323-8 (1977); Felton et al., Arch.Ophthalmol. 97 (9):1710-3 (1979); Lutty et al., Invest. Ophthalmol. Vis.Sci. 24 (1):52-6 (1983); Taylor and Weiss, Biochem Biophys Res Commun.133 (3):911-6 (1985); Zhu et al., Aust. N. Z. J. Ophthalmol. 25 Suppl1:S57-60 (1997); Ramesh et al., Br. J. Ophthalmol. 88 (5):697-702(2004)). Hyalocytes play also a role in the regression of the TVL(McMenamin et al., Invest. Ophthalmol. V is Sci. 43 (7):2076-82 (2002)).Lutty et al. have demonstrated that the hyalocytes produce and processtransforming growth factor-13 (TGF-(3) which may inhibit is theproliferation of the vascular endothelial cells in this system (Lutty etal., Invest. Ophthalmol. Vis. Sci. 34 (3):477-87 (1993)).

F. Several survival factors may protect cells from apoptosis, includingfibroblast growth factor (FGF), platelet-derived growth factor (PDGF)and vascular endothelial growth factor (VEGF). A reduction in levels ofthese growth factors below a critical threshold may lead to theinduction of an apoptotic program.

The role of VEGF in the maintenance of the VHP is not clear. Feenay etal. demonstrated that the TVL degeneration was unexpectedly uninfluencedby treatment with a VEGF A antibody, suggesting that programmedregression is independent of VEGF A, or that the development andmaturation of the lens had gone beyond the point of the plasticity andsusceptibility to certain growth factors (Feeney et al., Invest.Ophthalmol. Vis. Sci. 44 (2):839-47 (2003)).

SUMMARY

The present invention is based, at least in part, on the results ofproteomic analysis of the mouse lens and vitreous during postnataldevelopment, which identified novel proteins that contribute toregression of the hyaloid system. Among other proteins, two-dimensionalelectrophoresis and mass spectrometry of the developing mouse lens andvitreous identified activin receptor-like kinase-1 (ALK1) as adifferentially expressed protein during HVS regression;immunohistochemical staining demonstrated the localization of ALK1 inthe TVL; and overexpression of ALK1 in the cornea resulted in inhibitionof bFGF-induced corneal neovascularization in vivo. Activinreceptor-like kinase-1 (ALK1) activates Smads1, 5 and 8 whichdown-regulate VEGF production. In contrast, ALK5 activates Smads2 and 3which up-regulate VEGF production (Goumans et al., EMBO. J. 21,1743-1753 (2002)). Expression of ALK1 in blood vessels and mutations ofthe ALK1 gene in patients with human type II hereditary hemorrhagictelangiectasia, a multi-systemic vascular dysplasia, suggests that ALK1may play an important role during normal vascular development (Oh etal., Proc Natl Acad Sci USA 97, 2626-2631 (2000)).

Thus, ALK1 polypeptides and nucleic acids are useful compositions andtargets for treating disorders associated with neovascularization (NV),and in cancer therapy.

In one aspect, the invention provides methods for treating patients whohave a disorder associated with neovascularization, e.g., anophthalmological disorder associated with neovascularization. Themethods include administering to the patient a therapeutically effectiveamount of an ALK1 polypeptide or nucleic acid composition, e.g., asdescribed herein. The invention further includes therapeuticcompositions including the ALK1 polypeptides or nucleic acids describedherein, as well as inhibitors and/or agonists thereof, and methods foridentifying such compounds.

Ophthalmological disorders associated with neovascularization includeeye cancer, age-related macular degeneration, retinopathy ofprematurity, corneal graft rejection, glaucoma, diabetic retinopathy,wounds, age-related macular degeneration, herpetic and infectiouskeratitis, ocular ischemia, neovascular glaucoma, corneal, uveal andiris neovascularization, orbital and eyelid tumors, Stevens JohnsonSyndrome, ocular cicatricial pemphigoid, and ocular surface diseases. Insome embodiments, the ophthalmological disorder is associated withcorneal, retinal, choroidal, uveal, or iris neovascularization. For anophthalmological disorder, the administering can be, e.g., topical orparenteral administration into the eye, e.g., including, but not limitedto, local injection into or near the cornea, retina, vitreous, uvea,orbit, eyelid, conjunctiva, or iris.

In some embodiments, the disorder is cancer, e.g., as described herein.

In some embodiments, the subject is selected on the basis that he or shehas a disorder associated with neovascularization as described herein.

In some embodiments, the administering can be, e.g., local or systemic,e.g., parenteral or oral.

In another aspect, the invention provides methods for identifyingcandidate therapeutic compounds for the treatment of a disorderassociated with neovascularization. The methods include obtaining asample including a cell that is capable of expressing one or more of thegenes listed in Tables 1-6; contacting the sample with the testcompound; and evaluating levels, expression, or activity of the gene(s)in the sample, e.g., in the cell. Modulation of levels, expression, oractivity of the gene(s) in the sample indicates that the test compoundis a candidate therapeutic compound for the treatment of a disorderassociated with neovascularization. The levels, expression, or activityof the gene in the sample can be evaluated by methods known in the art,e.g., enzyme assays, immunoassays, high-throughput DNA, RNA, or proteinassays, etc. In some embodiments, the gene is ALK1.

In some embodiments, the method further includes administering thecandidate therapeutic compound to an animal model of a disorderassociated with neovascularization, and monitoring the animal model foran effect of the candidate therapeutic compound on a parameter of thedisorder, e.g., vascularisation, in the animal. A candidate therapeuticcompound that causes an improvement in the parameter in the animal modelis a candidate therapeutic agent for the treatment of the disorder. Insome embodiments, the methods further include administering thecandidate therapeutic agent to a subject having the disorder, e.g., asubject in a clinical trial, and monitoring a parameter of the disorderin the subject. A candidate therapeutic agent that improves theparameter in the subject is a therapeutic agent for the treatment of thedisorder. In some embodiments, the parameter is visual acuity. In someembodiments, the methods include administering a therapeuticallyeffective amount of the therapeutic agent to a subject in need oftreatment for the disorder, thereby treating the disorder.

In a further aspect, the invention provides methods for identifyingcandidate compounds, e.g., naturally-occurring compounds, e.g., apolypeptide or biologically active fragment thereof, for the treatmentof a disorder associated with neovascularization. The methods includeproviding a sample including:

-   -   (i) cells from a subject, e.g., a human subject, having a        disorder associated with blood vessel regression, e.g.,        retinopathy of prematurity, persistent hyperplastic primary        vitreous (PHPV), or retrolental fibroplasia; or    -   (ii) cells from a subject, e.g., a human subject, in a stage of        development that is associated with vessel regression, e.g., a        stage in the development of the hyaloid vascular system;        determining the expression levels of proteins, e.g., one or        more, e.g., 2, 3, 5, 10, 15, 20, 25, 30, 50 or 100, of the of        the proteins listed in Tables 1-6, in the sample; and comparing        the expression levels to a reference, e.g., a sample from an        unaffected subject or a subject at a different stage of        development associated with blood vessel regression, e.g., a        different stage the development of the hyaloid vascular system.        A compound that is differentially expressed, e.g., significantly        differentially expressed, is a candidate compound for the        treatment of a disorder associated with neovascularization.

In another aspect, the invention includes methods for treating adisorder associated with neovascularization, e.g., an ophthalmologicaldisorder associated with neovascularization as described herein, in asubject by administering a therapeutically effective amount of atherapeutic composition including an activin receptor-like kinase-1(ALK1) polynucleotide or polypeptide, or an active fragment thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C. Proteomic analysis of proteins from the lens and vitreous ofP1 and P16 mice. The merged image (1A) represents the warped imagebetween P1 (1B) and P16 (1C) obtained with Phoretix 2D evolutionsoftware. The proteins excised from 2-D gel (P16) for analysis andidentification by mass spectrometry are identified with numbers 1-20(1C).

FIGS. 2A-Q. bFGF-pellet induced corneal neovascularization (NV) isinhibited by naked ALK1 DNA injection in vivo. No-pellet controls areshown in 3A-H: Injection of naked DNA [ALK1 (2E-H) and vector only(2A-D)] did not induce corneal NV. The vector plus pellet positivecontrols are shown in 3I-L: development of NV in the corneal stroma wasevident by day 4 (2J); new vessels continued to grow in the direction ofthe pellet on days 7 and 14. None of the mice in the ALK1 and pelletgroups (2M-P) showed development of corneal NV on days 1, 4, 7, and 14after pellet implantation. Asterisk (*) indicates pellet implantation.The area of corneal NV of the four groups at days 1-14 is shown (2Q).

FIG. 3A-B are the amino acid (3A; SEQ ID NO:1) and nucleic acid (3B; SEQID NO:2) sequences of human ALK1.

DETAILED DESCRIPTION

A proteomic approach was used to decipher the biochemical eventscoexistent with the time points of the progressive regression of theHVS. Reproducible two-dimensional electrophoresis (2-DE) maps were usedto study modifications of the protein expression profiles occurring inthe mouse lens and vitreous at various stages of the regression of thehyaloid capillaries network. The identity of separated anddifferentially expressed proteins was confirmed by mass spectrometry(Thermo Electron's Finnigan LCQ Deca XP Plus™ Electrospray ionization(ESI) mass spectrometer).

In particular, attention was focused on the differential proteinexpression between postnatal day 1 and postnatal day 16. Opticalmicroscopy and hematoxylin-eosin staining studies showed the progressiveregression of the hyaloid vascular system of the mouse eye between P1,in which PM, TVL and HV are present and P16, in which these maturecapillary structures²¹ are almost completely regressed. Proteinsidentified in these screens are targets for therapeutic intervention forthe treatment of various disorders associated with neovascularization.Tables 1-6 list proteins which are expected to play a role invascularisation, inhibition of blood vessel regression, both in the HVSand in other systems, e.g., including pathophysiological processes, andare thus useful in the treatment of disorders associated withneovascularization, as described herein.

Activin Receptor-Like Kinase-1 (ALK1)

TGF-β is a potent inhibitor of vascular endothelial cell proliferation(Lutty et al., (1993), supra). It regulates endothelial proliferationvia two receptor/Smad (mother against decapentaplegic) pathways. Afterligand binding and activation of Type I receptors, signals aretransduced from the membrane to the nucleus via Smads (Seki et al., CircRes 93, 682-689 (2003)). Type I receptors recruit and phosphorylateSmads, such as Smads2 and 3 by the TβRI/ALK5 type I receptor in responseto TGF-β, and Smads1, 5 and 8 by the BMP (bone morphogenetic protein)type I receptors (Feng, Ann. Rev. Cell. Dev. Biol. 659-693 (2005)).

ALK1 is one of the seven Type I receptors for the TGF-β family ofproteins (ten Dijke et al., Science 264, 101-104 (1994); GeneID: 94;GenBank Accession Nos. NM_(—)000020.1 (nucleic acid) and NP_(—)000011.1(polypeptide)). ALK1 expression has been detected in endothelial cellsof highly vascularized tissues (lungs and placenta) (Panchenko et al.,Am. J. Physiol. 270, L547-L558 (1996)), normal and neoplastic pituitarycells (Alexander et al., J. Clin. Endocrinol. Metab. 81, 783-790(1996)), anaplastic large cell lymphoma (Sadahira et al., Pathol. Res.Pract. 195, 657-661 (1999)), inflammatory myofibroblastic tumor (Coffinet al., Mod. Pathol. 14, 569-576 (2001)) and central nervous systemcells (Pulford et al., Blood 89, 1394-1404 (1997)). ALK1 transduces theTGF-β1 signal by phosphorylating Smad1, Smad5 or Smad8 (Goumans, et al.,EMBO. J. 21, 1743-1753 (2002); Oh et al., Proc. Natl. Acad. Sci. U.S.A.97, 2626-2631 (2000)). In contrast, ALK5 activates Smads2 and 3 whichup-regulate VEGF production (Goumans et al., EMBO J. 21, 1743-1753(2002)). Upon phosphorylation by the receptors, Smad complexestranslocate into the nucleus, where they cooperate withsequence-specific transcription factors at the promoter DNA to regulategene expression (Feng and Derynck, Arum. Rev. Cell. Dev. Biol. 659-693(2005)). This functional and physical interaction confer bothspecificity and complexity in transcriptional responses to TGF-β familyligands (Id.).

Mutations of the ALK1 or endoglin genes have been linked to the humanvascular disorder Hereditary Hemorrhagic Telangiectasia (HHT) (Johnsonet al., Nat. Genet. 13, 189-195 (1996); McAllister et al., Nat. Genet.8, 345-351 (1994)). This is an autosomal-dominant disorder, also knownas Osler-Rendu-Weber syndrome, characterized by the age-dependentdevelopment of focal arteriovenous malformations and telangiectases(Srinivasan et al., Hum. Mol. Genet. 12, 473-482 (2003)). HHT type 2 iscaused by loss of function of the activin receptor-like kinase 1 (ACVRL1or ALK1) (Id.). The disease is characterized by dilated, thin-walled,vascular anomalies of the skin and mucous membranes and recurrentepistaxis. In the literature, abnormal eye disorders have beendocumented in 45-□65% of patients with HHT, with the most common lesionsbeing conjunctival telangiectasias (Brant et al., Am. J. Ophthalmol.107, 642-646 (1989); Vase and Vase, Acta. Ophthalmol. Copen. 57,1084-1090 (1979)). Retinal arteriovenous malformations, retinaltelangiectasia and choroidal hemorrhage during intraocular surgery havealso been noted (Brant et al., (1989) supra; Mahmoud et al., Am. J.Ophthalmol. 133, 282-284 (2002)).

The sequences of the human ALK1 polypeptide (SEQ ID NO:1) andpolynucleotide (SEQ ID NO:2) are shown in FIG. 3. Active fragmentsthereof include, e.g., amino acids 22-503 of SEQ ID NO:1, or nucleotides346-1791 of SEQ ID NO:2. Active fragments can also include the GS motif,i.e., amino acids 173-202 of SEQ ID NO:1; the Serine/Threonine proteinkinases, catalytic domain, i.e., amino acids 204-487 of SEQ ID NO:1;and/or the Activin types I and II receptor domain, i.e., amino acids19-103 of SEQ ID NO:1. An active fragment retains the ability to blockbFGF-induced neovascularization. Homologs of the human ALK1 in otherspecies are known, e.g., for rat, GenBank Accession No. NM_(—)022441.1;for mouse, NM_(—)009612.1

Pharmaceutical Compositions and Methods of Administration

The polypeptides listed in Tables 1-6, and nucleic acid moleculesencoding or inhibiting them, can be incorporated into pharmaceuticalcompositions as active ingredients. In some embodiments, thepharmaceutical compositions include a human ALK1 polypeptide (e.g., SEQID NO:1) or polynucleotide (e.g., SEQ ID NO:2), or an active fragmentthereof. In general, it will be preferable to match the composition tothe species that is being treated; thus, for example, when treatingexperimental animals such as mice other species can be used, e.g., fromrats or mice.

Pharmaceutical compositions typically include the active ingredient anda pharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition is typically formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, the composition is especially adapted foradministration into or around the eye. For example, a composition can beadapted to be used as eye drops, or injected into the eye, e.g., usingperibulbar or intravitreal injection. Such compositions should besterile and substantially endotoxin-free, and within an acceptable rangeof pH. Certain preservatives are thought not to be good for the eye, sothat in some embodiments a non-preserved formulation is used.Formulation of eye medications is known in the art, see, e.g., OcularTherapeutics and Drug Delivery: A Multi-Disciplinary Approach, Reddy,Ed. (CRC Press 1995); Kaur and Kanwar, Drug Dev Ind Pharm. 2002 May;28(5):473-93; Clinical Ocular Pharmacology, Bartlett et al.(Butterworth-Heinemann; 4th edition (Mar. 15, 2001)); and OphthalmicDrug Delivery Systems (Drugs and the Pharmaceutical Sciences: a Seriesof Textbooks and Monographs), Mitra (Marcel Dekker; 2nd Rev&Ex edition(Mar. 1, 2003)).

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Administration of a therapeutic composition described herein can also beby transmucosal or transdermal means. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, for example, for transmucosal administration,detergents, bile salts, and fusidic acid derivatives. Transmucosaladministration can be accomplished through the use of nasal sprays orsuppositories. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

Compositions comprising nucleic acid compounds can also be administeredby any method suitable for administration of nucleic acid agents. Thesemethods include gene guns, bio injectors, and skin patches as well asneedle-free methods such as the micro-particle DNA vaccine technologydisclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermalneedle-free vaccination with powder-form vaccine as disclosed in U.S.Pat. No. 6,168,587. Additionally, intranasal delivery is possible, asdescribed in, inter alia, Hamajima et al., Clin. Immunol. Immunopathol.,88(2), 205-10 (1998). Liposomes (e.g., as described in U.S. Pat. No.6,472,375) and microencapsulation can also be used. Biodegradabletargetable microparticle delivery systems can also be used (e.g., asdescribed in U.S. Pat. No. 6,471,996). In some embodiments, the nucleicacid compounds comprise naked DNA, and are administered locally byinjection, e.g., as described herein.

In some embodiments, the compositions are prepared with carriers thatwill protect the active ingredient against rapid elimination from thebody, such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. The materialscan also be obtained commercially, e.g., from Alza Corporation or NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

Dosage, toxicity and therapeutic efficacy of the compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the 1050 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. For example, a therapeutic amount is one that achievesa desired therapeutic effect. This amount can be the same or differentfrom a prophylactically effective amount, which is an amount necessaryto prevent onset of disease or disease symptoms. An effective amount canbe administered in one or more administrations, applications or dosages.A therapeutically effective amount of a composition depends on thecomposition selected. The compositions can be administered one from oneor more times per day to one or more times per week; including onceevery other day. The skilled artisan will appreciate that certainfactors may influence the dosage and timing required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of the compositions ofthe invention can include a single treatment or a series of treatments.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Nucleic Acids for Expression

The therapeutic compositions described herein can include nucleic acidmolecules encoding a selected protein, e.g., a protein listed in one ofTables 1-6, e.g., ALK1; these are useful, e.g., where an increase in theexpression and/or activity of a selected protein is desirable. Nucleicacid molecules comprising expression constructs can be used, e.g., forin vivo or in vitro expression of a selected protein. In someembodiments, expression can be restricted to a particular cell types soas to reconstitute the function of the selected protein in a cell, e.g.,a cell in which that polypeptide is misexpressed, or in which expressionof that polypeptide would produce a therapeutic benefit.

A nucleic acid encoding the selected protein can be inserted in anexpression vector, to make an expression construct. A number of suitablevectors are known in the art, e.g., viral vectors including recombinantretroviruses, adenovirus, adeno-associated virus, and herpes simplexvirus-1, adenovirus-derived vectors, or recombinant bacterial oreukaryotic plasmids. For example, the expression construct can include:a coding region; a promoter sequence, e.g., a promoter sequence thatrestricts expression to a selected cell type, a conditional promoter, ora strong general promoter; an enhancer sequence; untranslated regulatorysequences, e.g., a 5′ untranslated region (UTR), a 3′UTR; apolyadenylation site; and/or an insulator sequence. Such sequences areknown in the art, and the skilled artisan would be able to selectsuitable sequences. See, e.g., Current Protocols in Molecular Biology,Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989),Sections 9.10-9.14, and other standard laboratory manuals.

Expression constructs can be administered in any biologically effectivecarrier, e.g. any formulation or composition capable of effectivelydelivering the component gene to cells in vivo. Viral vectors transfectcells directly; plasmid DNA can be delivered with the help of, forexample, cationic liposomes (e.g., Lipofectin) or derivatized (e.g.antibody conjugated), polylysine conjugates, gramicidin S, artificialviral envelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation. In someembodiments, the nucleic acid is applied “naked” to a cell, i.e., isapplied in a simple buffer without the use of any additional agents toenhance uptake. See, e.g., Current Protocols in Molecular Biology,Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989),Sections 9.10-9.14 and other standard laboratory manuals.

In clinical settings, the nucleic acids can be introduced into a subjectby any of a number of methods, each of which is familiar in the art. Forinstance, a pharmaceutical preparation of the gene delivery system canbe introduced systemically, e.g. by intravenous injection, and specifictransduction of the protein in the target cells occurs predominantlyfrom specificity of transfection provided by a targeted gene deliveryvehicle, cell-type or tissue-type expression due to the transcriptionalregulatory sequences controlling expression of the receptor gene, or acombination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle can beintroduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (e.g. Chen et al. (1994) PNAS 91: 3054-3057).

Inhibitory Nucleic Acid Molecules

The therapeutic compositions described herein can include inhibitorynucleic acid molecules that are targeted to a selected target RNAencoding a protein listed in one of Tables 1-6, e.g., antisense, siRNA,ribozymes, and aptamers; these are useful, e.g., where a decrease in theexpression and/or activity of a target protein is desirable. Based uponsequences known in the art, one of skill in the art can easily chooseand synthesize any of a number of appropriate inhibitory nucleic acidmolecules for use in accordance with the present invention. For example,a “gene walk” comprising a series of oligonucleotides of 15-30nucleotides spanning the length of a target nucleic acid can beprepared, followed by testing for inhibition of target gene expression,to provide an antisense sequence. Optionally, gaps of 5-10 nucleotidescan be left between the oligonucleotides to reduce the number ofoligonucleotides synthesized and tested. Similar methods can be used togenerate siRNAs, aptamers, and ribozymes

siRNA Molecules

RNAi is a process whereby double-stranded RNA (dsRNA, also referred toherein as si RNAs or ds siRNAs, for double-stranded small interferingRNAs,) induces the sequence-specific degradation of homologous mRNA inanimals and plant cells (Hutvagner and Zamore, Curr. Opin. Genet.Dev.:12, 225-232 (2002); Sharp, Genes Dev., 15:485-490 (2001)). Inmammalian cells, RNAi can be triggered by 21-nucleotide (nt) duplexes ofsmall interfering RNA (siRNA) (Chiu et al., Mol. Cell. 10:549-561(2002); Elbashir et al., Nature 411:494-498 (2001)), or by micro-RNAs(miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which areexpressed in vivo using DNA templates with RNA polymerase III promoters(Zeng et al., Mol. Cell. 9:1327-1333 (2002); Paddison et al., Genes Dev.16:948-958 (2002); Lee et al., Nature Biotechnol. 20:500-505 (2002);Paul et al., Nature Biotechnol. 20:505-508 (2002); Tuschl, T., NatureBiotechnol. 20:440-448 (2002); Yu et al., Proc. Natl. Acad. Sci. USA99(9):6047-6052 (2002); McManus et al., RNA 8:842-850 (2002); Sui etal., Proc. Natl. Acad. Sci. USA 99(6):5515-5520 (2002).)

The nucleic acid molecules or constructs can include dsRNA moleculescomprising 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleotides in each strand, wherein one of the strands issubstantially identical, e.g., at least 80% (or more, e.g., 85%, 90%,95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatchednucleotide(s), to a target region in the mRNA, and the other strand iscomplementary to the first strand. The dsRNA molecules can be chemicallysynthesized, or can transcribed be in vitro from a DNA template, or invivo from, e.g., shRNA. The dsRNA molecules can be designed using anymethod known in the art; a number of algorithms are known, and arecommercially available. Gene walk methods can be used to optimize theinhibitory activity of the siRNA.

The inhibitory nucleic acid compositions can include both siRNA andmodified siRNA derivatives, e.g., siRNAs modified to alter a propertysuch as the pharmacokinetics of the composition, for example, toincrease half-life in the body, as well as engineered RNAi precursors.

siRNAs can be delivered into cells by methods known in the art, e.g.,cationic liposome transfection and electroporation. siRNA duplexes canbe expressed within cells from engineered RNAi precursors, e.g.,recombinant DNA constructs using mammalian Pol III promoter systems(e.g., H1 or U6/snRNA promoter systems (Tuschl (2002), supra) capable ofexpressing functional double-stranded siRNAs; (Bagella et al., J. Cell.Physiol. 177:206-213 (1998); Lee et al. (2002), supra; Miyagishi et al.(2002), supra; Paul et al. (2002), supra; Yu et al. (2002), supra; Suiet al. (2002), supra). Transcriptional termination by RNA Pol III occursat runs of four consecutive T residues in the DNA template, providing amechanism to end the siRNA transcript at a specific sequence. The siRNAis complementary to the sequence of the target gene in 5′-3′ and 3′-5′orientations, and the two strands of the siRNA can be expressed in thesame construct or in separate constructs. Hairpin siRNAs, driven by H1or U6 snRNA promoter and expressed in cells, can inhibit target geneexpression (Bagella et al. (1998), supra; Lee et al. (2002), supra;Miyagishi et al. (2002), supra; Paul et al. (2002), supra; Yu et al.(2002), supra; Sui et al. (2002) supra). Constructs containing siRNAsequence under the control of T7 promoter also make functional siRNAswhen cotransfected into the cells with a vector expression T7 RNApolymerase (Jacque (2002), supra).

Antisense

An “antisense” nucleic acid can include a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to a TEF mRNA sequence. The antisense nucleic acid can becomplementary to an entire coding strand of a target sequence, or toonly a portion thereof. In another embodiment, the antisense nucleicacid molecule is antisense to a “noncoding region” of the coding strandof a nucleotide sequence (e.g., the 5′ and 3′ untranslated regions).

An antisense nucleic acid can be designed such that it is complementaryto the entire coding region of a target mRNA, but can also be anoligonucleotide that is antisense to only a portion of the coding ornoncoding region of the target mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of the target mRNA, e.g., between the −10 and +10regions of the target gene nucleotide sequence of interest. An antisenseoligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid can be constructed using chemical synthesisand enzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid (e.g., an antisense oligonucleotide)can be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. The antisense nucleic acid also can be produced biologically usingan expression vector into which a nucleic acid has been subcloned in anantisense orientation (i.e., RNA transcribed from the inserted nucleicacid will be of an antisense orientation to a target nucleic acid ofinterest, described further in the following subsection).

In some embodiments, the antisense nucleic acid molecule is an∀-anomeric nucleic acid molecule. ∀-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al., Nucleic Acids. Res. 15:6625-6641 (1987)). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. Nucleic Acids Res. 15:6131-6148(1987)) or a chimeric RNA-DNA analogue (Inoue et al. FEBS Lett.,215:327-330 (1987)).

In some embodiments, the antisense nucleic acid is a morpholinooligonucleotide (see, e.g., Heasman, Dev. Biol. 243:209-14 (2002);Iversen, Curr. Opin. Mol. Ther. 3:235-8 (2001); Summerton, Biochim.Biophys. Acta. 1489:141-58 (1999).

Target gene expression can be inhibited by targeting nucleotidesequences complementary to a regulatory region (e.g., promoters and/orenhancers) to form triple helical structures that prevent transcriptionof the Spt5 gene in target cells. See generally, Helene, Anticancer DrugDes. 6:569-84 (1991); Helene, Ann. N.Y. Acad. Sci. 660:27-36 (1992); andMaher, Bioassays 14:807-15 (1992). The potential sequences that can betargeted for triple helix formation can be increased by creating a socalled “switchback” nucleic acid molecule. Switchback molecules aresynthesized in an alternating 5′-3′,3′-5′ manner, such that they basepair with first one strand of a duplex and then the other, eliminatingthe necessity for a sizeable stretch of either purines or pyrimidines tobe present on one strand of a duplex.

Ribozymes

Ribozymes are a type of RNA that can be engineered to enzymaticallycleave and inactivate other RNA targets in a specific,sequence-dependent fashion. By cleaving the target RNA, ribozymesinhibit translation, thus preventing the expression of the target gene.Ribozymes can be chemically synthesized in the laboratory andstructurally modified to increase their stability and catalytic activityusing methods known in the art. Alternatively, ribozyme genes can beintroduced into cells through gene-delivery mechanisms known in the art.A ribozyme having specificity for a target nucleic acid can include oneor more sequences complementary to the nucleotide sequence of a cDNAencoding a protein described herein, and a sequence having knowncatalytic sequence responsible for mRNA cleavage (see U.S. Pat. No.5,093,246 or Haselhoff and Gerlach Nature 334:585-591 (1988)). Forexample, a derivative of a Tetrahymena L-19 WS RNA can be constructed inwhich the nucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a target mRNA. See, e.g., Cech etal. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, a target mRNA can be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See, e.g., Bartel and Szostak, Science 261:1411-1418 (1993).

Administration of Nucleic Acid Molecules

The nucleic acid molecules described herein can be administered to asubject (e.g., by direct injection at a tissue site), or generated insitu. For example, inhibitory nucleic acid molecules can be administeredsuch that they hybridize with or bind to cellular mRNA and/or genomicDNA encoding a target protein to thereby inhibit expression of theprotein, e.g., by inhibiting transcription and/or translation. Nucleicacid molecules that encode an active agent are generally administeredsuch that they enter a cell and are expressed therein.

In some embodiments, the nucleic acid molecules can be administeredselectively, e.g., by local injection, or modified to target selectedcells (e.g., by the use of a tissue-specific promoter) and thenadministered systemically. For systemic administration, nucleic acidmolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface, e.g., by linking thenucleic acid nucleic acid molecules to peptides or antibodies that bindto cell surface receptors or antigens. The nucleic acid nucleic acidmolecules can also be delivered to cells using vectors known in the art,e.g., as described herein. To achieve sufficient intracellularconcentrations of the inhibitory nucleic acid molecules, vectorconstructs in which the inhibitory nucleic acid nucleic acid molecule isplaced under the control of a promoter, e.g., a strong promoter can beused. Methods for creating suitable vectors, and choosing promoters, areknown in the art.

Methods of Treatment

The polypeptides and nucleic acids described herein, e.g., ALK1polypeptides (e.g., SEQ ID NO:1) or polynucleotides (e.g., SEQ ID NO:2),and active fragments thereof, are useful in the treatment of disordersassociated with neovascularization, i.e., abnormal angiogenic processes,e.g., disorders in the formation of blood vessels. Typically, thedisorder will stem from overformation of blood vessels, or formation ofblood vessels in an unwanted area, e.g., in the avascular regions of theeye, e.g., retinopathies, or in a tumor, e.g., a cancerous or benigntumor. For example, the ophthalmological disorder can be age-relatedmacular degeneration, where new blood vessels grow under the retina, ordiabetic retinopathy, where abnormal vessels grow on top of the retina.Other ophthalmological disorders include retinopathy of prematurity,corneal graft rejection, glaucoma, herpetic and infectious keratitis,ocular ischemia, neovascular glaucoma, corneal, uveal and irisneovascularization, orbital and eyelid tumors, Stevens Johnson Syndrome,ocular cicatricial pemphigoid, wounds, and ocular surface diseases. Thedisorder can be characterized by, for example, corneal, retinal,choroidal, uveal, or iris neovascularization

The disorder may stem from the formation of blood vessels that deliverblood to a tissue, e.g., a primary or metastatic cancerous or benigntumors, e.g., cancer. A metastatic tumor can arise from a multitude ofprimary tumor types, including but not limited to those of prostate,colon, lung, breast and liver origin.

As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair; thus, the methods include administrationof a compound identified by a method described herein to maintain avascularity during wound healing. In this embodiment, theophthalmological disorder is a wound, including both accidental as wellas intentional wounds (e.g., surgical wounds).

In some embodiments, the ophthalmological disorder is a cancer of theeye, e.g., eyelid tumors, e.g., malignant eye lid tumors, benign eye lidtumors, basal cell carcinoma, squamous cell carcinoma, sebaceous cellcarcinoma, and malignant melanoma; conjunctival tumors, e.g., pigmentedconjunctival tumors, melanoma and primary acquired melanosis withatypia, squamous conjunctival neoplasia, conjunctival lymphoma, andKaposi's Sarcoma; iris tumors, e.g., iris melanoma, iris pigmentepithelial cyst, anterior uveal metastasis, and pearl cyst of the iris;infiltrative intraocular tumors, e.g., multiple myeloma, lymphoma, andleukemia; choroidal tumors, e.g., choroidal melanoma, choroidalmetastasis, choroidal nevus, choroidal hemangioma, choroidal osteoma,and Nevus of Ota; retinal tumors, e.g., retinoblastoma, retinal pigmentepithelial tumors, retinal pigment epithelial hypertrophy, von Hippelangioma; optic nerve tumors, e.g., melanocytoma, melanoma, meningioma,circumpapillary metastasis; orbital tumors, e.g., lymphangioma,cavernous hemangioma, meningioma, mucocele, rhabdomyosarcoma, orbitalpseudotumor, adenoid cystic carcinoma, periocular hemangioma ofchildhood; cancers of the ocular adnexa, e.g., lacrimal gland carcinomassuch as adenoid cystic carcinoma and mucoepidermal epithelioma; andmetastatic ocular tumors, e.g., metastatic choroidal melanoma, andmetastatic retinoblastoma.

As used in this context, to “treat” means to ameliorate at least onesymptom associated with abnormal angiogenesis as well as reduceneovascularization. For the treatment of cancers and solid tumors, to“treat” includes inhibition of the growth of blood vessels resulting ina lack of nutrients for the tumors and/or cancer cells needed by thetumor for its growth. Tumors and growths will decrease in size andpossibly disappear. Administration of a therapeutically effective amountof a composition for the treatment of arthritic conditions will resultin decreased blood vessel formation in cartilage, specifically joints,resulting in increased mobility and flexibility in these regions. Inophthalmologic conditions, administration of a therapeutically effectiveamount of a composition described herein will reduce the formation ofextraneous blood vessels in the retina, resulting in unobstructed orless obstructed vision. In the treatment of disorders such as cancer,administration of a therapeutically effective amount of a compositiondescribed herein will inhibit the growth and/or further formation ofblood vessels, thereby inhibiting the formation of any lesions and/ortumors that arise.

Methods of Screening

The invention includes methods for screening test compounds, e.g.,polypeptides, polynucleotides, inorganic or organic large or smallmolecule test compounds, to identify agents useful in the treatment ofdisorders associated with neovascularization, e.g., as described herein.

As used herein, “small molecules” refers to small organic or inorganicmolecules of molecular weight below about 3,000 Daltons. In general,small molecules useful for the invention have a molecular weight of lessthan 3,000 Daltons (Da). The small molecules can be, e.g., from at leastabout 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 toabout 500 Da, about 200 to about 1500, about 500 to about 1000, about300 to about 1000 Da, or about 100 to about 250 Da).

The test compounds can be natural products or members of a combinatorialchemistry library. A set of diverse molecules can be used to cover avariety of functions such as charge, aromaticity, hydrogen bonding,flexibility, size, length of side chain, hydrophobicity, and rigidity.Combinatorial techniques suitable for synthesizing small molecules areknown in the art, e.g., as exemplified by Obrecht and Villalgordo,Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries, Pergamon-Elsevier ScienceLimited (1998), and include those such as the “split and pool” or“parallel” synthesis techniques, solid-phase and solution-phasetechniques, and encoding techniques (see, for example, Czarnik, Curr.Opin. Chem. Bio. 1:60-6 (1997)). In addition, a number of libraries,e.g., small molecule libraries, are commercially available. A number ofsuitable small molecule test compounds are listed in U.S. Pat. No.6,503,713, incorporated herein by reference in its entirety.

Libraries screened using the methods of the present invention cancomprise a variety of types of test compounds. A given library cancomprise a set of structurally related or unrelated test compounds. Insome embodiments, the test compounds are peptides or peptidomimeticmolecules. In some embodiments, the test compounds are nucleic acids,e.g., antisense, RNAi, or aptamers.

In some embodiments, the test compounds and libraries thereof can beobtained by systematically altering the structure of a first testcompound, e.g., a first test compound that is structurally similar to aknown natural binding partner of the target polypeptide, or a firstsmall molecule identified as capable of binding the target polypeptide,e.g., using methods known in the art or the methods described herein,and correlating that structure to a resulting biological activity, e.g.,a structure-activity relationship study. As one of skill in the art willappreciate, there are a variety of standard methods for creating such astructure-activity relationship. Thus, in some instances, the work maybe largely empirical, and in others, the three-dimensional structure ofan endogenous polypeptide or portion thereof can be used as a startingpoint for the rational design of a small molecule compound or compounds.For example, in one embodiment, a general library of small molecules isscreened, e.g., using the methods described herein.

In some embodiments, a test compound is applied to a test sample, e.g.,a cell or living tissue or organ, e.g., an eye, and one or more effectsof the test compound is evaluated. In a cultured or primary cell forexample, the ability of the test compound to modulate expression of oneor more of the proteins listed in Tables 1-6 can be evaluated. In theeye, for example, the ability of the test compounds to modulateexpression of one or more of the proteins listed in Tables 1-6 can beevaluated.

Methods for evaluating each of these effects are known in the art. Forexample, ability to modulate expression of a protein can be evaluated atthe gene or protein level, e.g., using quantitative PCR or immunoassaymethods. In some embodiments, high throughput methods, e.g., protein orgene chips as are known in the art (see, e.g., Ch. 12, Genomics, inGriffiths et al., Eds., Modern Genetic Analysis, 1999, W.H. Freeman andCompany; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218;MacBeath and Schreiber, Science 2000, 289(5485):1760-1763; Simpson,Proteins and Proteomics: A Laboratory Manual, Cold Spring HarborLaboratory Press; 2002; Hardiman, Microarrays Methods and Applications:Nuts & Bolts, DNA Press, 2003), can be used to detect an effect on two,three, four, five or more of the proteins listed in Tables 1-6.

Compounds identified as “hits” (e.g., test compounds that demonstrate anability to modulate, i.e., cause an increase or decrease in levels,expression, or activity, of one or more of the proteins listed in Tables1-6) in the first screen can be selected and systematically altered,e.g., using rational design, to optimize binding affinity, avidity,specificity, or other parameter. Such optimization can also be screenedfor using the methods described herein. Thus, in one embodiment, theinvention includes screening a first library of small molecules using amethod known in the art and/or described herein, identifying one or morehits in that library, subjecting those hits to systematic structuralalteration to create a second libraries of compounds structurallyrelated to the hit, and screening the second library using the methodsdescribed herein.

Small molecules identified as hits can be considered candidatetherapeutic compounds, useful in treating disorders associated withneovascularization, as described herein. A variety of techniques usefulfor determining the structures of “hits” can be used in the methodsdescribed herein, e.g., NMR, mass spectrometry, gas chromatographyequipped with electron capture detectors, fluorescence and absorptionspectroscopy. Thus, the invention also includes compounds identified as“hits” by the methods described herein, and methods for theiradministration and use in the treatment, prevention, or delay ofdevelopment or progression of a disorder described herein.

Test compounds identified as candidate therapeutic compounds can befurther screened by administration to an animal model of a disordersassociated with neovascularization, as described herein. The animal canbe monitored for a change in the disorder, e.g., for an improvement in aparameter of the disorder, e.g., a parameter related to clinicaloutcome. In some embodiments, the parameter is unwanted vascularisation,and an improvement would be a decrease in the levels of vascularisation.

One of skill in the art will appreciate that the methods describedherein can be performed on non-rodent, e.g., human samples, e.g.,samples obtained from human fetuses at different stages of development,as well as samples at substantially the same stage of development, butin pathological and non-pathological stages. For example, a surgicalspecimen from a subject with a disorder, e.g., retinopathy ofprematurity or retrolental fibroplasias, can be used, and compared witha normal, unaffected control, e.g., an age-matched control.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Observation of Whole-Mount Specimens

To document the changes in the hyaloid vascular system associated withdevelopment in the cornea, whole-mount specimens were observed.

A total of 44 C57BL/6 newborn male and female litters were obtained fromJackson Laboratories (Bar Harbor, Me.) were used for the experimentsdescribed herein. The age of the newborn mice was expressed in terms ofpost-gestational days. The newborn mice were sacrificed onpost-gestational days 1, 4, 8, 16, 24, and 30 (i.e., P1, p4, p16, p24,and P30). Four eyes per each post-gestational day were used for thehistological study. For the lens and vitreous protein extraction wereused 20 eyes of P 1 mice, 20 eyes of P 4 mice, 12 eyes of P 8 mice and12 eyes of P 16 mice. Each of the 12 newborn mice used for thehistological study was perfused with 4% paraformaldehyde in PBS undergeneral anesthesia administered by intramuscular injection of a mixtureof ketamine (200 mg/kg) and xylazine (10 mg/kg). The eyes were removedand immerse in buffered neutral formalin solution (100 ml 40% formalin,900 ml distilled water, 4.0 gm monobasic sodium phosphate, 6.5 gmdibasic sodium phosphate), then they were embedded into paraffin andwere cut in tissue slices of 5 micron each with a microkeratome. Eachsample was stained with Hematoxylin-Eosin (H-E) staining.

By embryonic day 11 (E11) the hyaloid artery enters the optic cup andextends anteriorly, branching to form the vascular tunic of the lens(Smith, Systematic evaluation of the Mouse Eye: Anatomy, Pathology, andBiomethods. Pp 45-63. Sunders, 2002.). From E14 to birthday the bloodvessels that occupied most of the vitreous cavity in earlier stages havebecome less prominent except around the lens and along the inner retinalsurface; the PM and the TVL remain prominent (Smith, Systematicevaluation of the Mouse Eye: Anatomy, Pathology, and Biomethods. Pp45-63. Sunders, 2002.).

PM

The PM of the newborn mice showed as a dense plexus of capillaries thatbegan to regress on post-gestation day 4 and become vestigial on day 8.The PM disappeared completely in all eyes on day 16.

TVL

The radial branches of the TVL that lie on the lens surface areprominent until P4, particularly posterior to the lens equator. Laterthere is a gradual disappearance of the TVL and it is completelydisappeared in about one third of eyes examined on day P24 and in alleyes on day P30.

HA and VHP

The HA arose from the optic disc as a single thick proximal trunk andentered the vitreous branching in several vessels of the VHP and runninganteriorly to the posterior pole of the lens. As the whole eye developedin size and the volume of the vitreous increased, the VHP run toward thelens joining the TVL and became less prominent. Involution of HA and VHPhas begun by day 1 and from day 12 to day 30 there is a gradualdisappearance of those vessels.

These results concur with previous reports of HVS development andregression.

Example 2 Progressive Regression of HVS in the Mouse and DifferentialProtein Expression Profile

This example describes the use of a proteomic approach to try todecipher the biochemical events coexisting with the progressiveregression of the HVS. The proteomic analysis provided an opportunity tocompare different stages of the mouse eye development, to identifyspecific qualitative and quantitative protein changes, associated withthe early maintenance and later regression of the HVS.

Therefore to fully characterize and analyze such a dynamic process,experiments should be performed that capture the protein expressionprofiles of the lens and the vitreous at the various stages of bloodvessel regression.

To prepare mouse lens and vitreous for 2-D electrophoresis, 32 newbornmice were decapitated. The eyes were enucleated and then frozen at −80degrees Celsius. The frozen eyes were scraped with a blade to removecornea, aqueous humor, conjunctiva, sclera, iris, ciliary body, uvea andretina and to obtain the lens surrounded by PM and TVL and the primaryvitreous containing VHP.

The scraping was done on a dry-ice bed to prevent the specimen fromwarming up and the consequent protein denaturation, melting of thetissue and contamination of the specimen by eye components differentfrom lens and primary vitreous.

Twenty specimens of P1, 20 specimens of P4, 12 specimens of P8, and 12specimens of P16 were independently solubilized in 250 μl of TotalProtein Extraction Buffer (7 M urea, 2 M thiourea, 1% (w/v) ASB-14detergent, 40 mM Tris base, 0.001% bromophenol blue, 20% carrierampholyte; Biorad Laboratories, Hercules, Calif.) plus 2 of 200 mMtributylphosphine by mechanically homogenizing them with an electricaltissue homogenizer for 5 minutes, in an ice-water bath to prevent thespecimens from warming up.

The homogenate was transferred into 1.5 ml micro centrifuge tubes andthe protein extracts were cleared by centrifugation at 14,000 rpm for 20minutes at 4° Celsius to remove particulates. The protein concentrationof the cleared supernatants was determined by using a compatible proteinassay (Biorad Laboratories, Hercules, Calif.), and samples were storedat −80° Celsius until use.

Three separate experiments were performed.

I. Differential Protein Expression in P1, P4, P8, P16 Using IPG stripspH 3-10

A time course experiment was designed to examine the changes in proteinexpression at the critical time points throughout the HVS regressionprocess.

Lens and vitreous of P1, P4, P8 and P16 were selected to perform theexperiment because the histological analysis showed that inpost-gestation day 1 the hyaloid network is still prominent and bypost-gestational day 16 the hyaloid network is already vestigial. Inthis experiment, three samples per each post-gestational day were loadedonto nonlinear immobilized pH gradient (IPG) gel strips (7 cm, pH 3-10)and rehydrated overnight with Rehydration/Sample Buffer (BioradLaboratories, Hercules, Calif.).

As this is the first reported application of proteome analysis on HVSregression, several of the conditions for tissue selection andpreparation were developed and optimized to obtain clean specimen oflens and vitreous to avoid the presence of proteins coming from otherpart of the eye on the final 2-DE gels.

Images of the 2-DE gels were captured with Molecular Imager FX Pro Plusmulti-imager system and protein expression profile at each time pointwas compared in triplicate using the Phoretix 2D imaging analysissoftware.

Protein spots that were reproducibly differentially expressed in P1 andP 16 were considered for protein identification. The statisticalsignificance of changes was evaluated using the Phoretix 2D software.

Gel bands were excised and minced into approximate 1 mm³ pieces with asterile razor blade of the Xcise technology platform from Proteomesystem and placed into a sterile microcentrifuge. Gel pieces weredestained 3 times by adding 200 uL of 25 mM NH₄HCO₃ in 50% acetonitrile,dried using a SpeedVac® concentrator, alkylated and digested withtrypsin (Promega, Madison, Wis., USA) in 50 mM of ammonium bicarbonateovernight to release tryptic peptides. Samples were dried in a SpeedVac®to remove residual ammonium bicarbonate. Peptides were resuspended in50% acetonitrile with 1% formic acid solution. About 30% of the digestwas subjected to nano-LC ESI IT MS/MS analysis.

A Surveyor LC pump (ThermoElectron, Calif.) with a C18 trapping column(300 um i.d.×1 mm, Dionex, Calif.) and a self-packed reversed-phasecolumn 75 mm i.d.×15 cm (Magic C18AQ, 3 um) was used for nano-LCexperiments. An LCQ Deca XP Plus ESI mass spectrometer (Thermo Electron,Calif.) was used for all the experiments. In data-dependent MS/MSscanning, a full MS scan between 400 and 2000 m/z was followed by fivefull MS/MS scans for the five most intense ions from the MS scan in allESI. MS/MS data-dependent acquisition, followed by database searchingwith SEQUEST (BioWorks 3.1, ThermoElectron) allowed proteinidentification.

Fully tryptic peptides were matched with SEQUEST at a delta correlation( )Cn) of greater than 0.08 and correlation (Xcorr) greater than 1.9,2.2 and 3.5 for charged states of +1, +2 and +3, respectively. Thesearch was performed against the whole NCBInr mouse protein sequencedatabase, and human homologs (e.g., proteins with at least 75% identityto the mouse sequence) were identified (www.ncbi.nml.nih.gov).

Protein expression maps were generated in triplicate per each time pointobtaining a reproducible separation of the protein spots on the 2-DEgels. On average, up to 1300 proteins spots could be detected in eachgel with the Phoretix 2D imaging analysis software. This softwareprovides the ability to “warp” different gel images using mathematicalalgorithms, improving the quality of the protein spot matching betweenthe gels and allowing easy detection of the differences among the gels.

The 2-DE gels demonstrated a progressive lessening in the number and inthe intensity of the protein spots from P1 to P16, particularly of theproteins that migrated in the area corresponding to pI 4 to pI 7 andM_(r) 30 kDa to 90 kDa. However, the warping of P1, P4 and P8 with P16revealed the presence of 20 protein spots in P16 that are not present inthe other gels.

This evidence suggest the presence in the earlier post-gestation days ofan active protein expression coexisting with the presence of a prominentHVS and the presence in P16 of factors whose synthesis or degradation isrelated to the regression of the HVS itself.

For this initial study, 11 protein spots were identified, using thePhoretix 2D imaging analysis software, that were present in P1 atgreater than two-fold higher levels of expression relative to P16 wereselected and excised for identification. From these 11 spots, 23differentially expressed proteins were identified (Table 1); one of themis an unnamed protein product and two of them (the serotransferrinprecursor and similar to ethanol induced 6) were identified in multiplespots, as they are post-translationally modified versions of the sameprotein.

TABLE 1 Proteins Present in P1 at >2X levels of expression in P16 MOUSEHUMAN GENE HOMOLOG # PROTEIN BANK # GENE BANK # 1. alpha fetoprotein31982513  120042 2. Calmodulin 115512 56404656 3. protein product19263784 19263784 (—) 4. Creatine kinase 15929689 34335231 5.dermatan-4- 31980715 18497304 sulfotransferase-1 6. Ethanol induced 6homolog 38082607 (—) 7. GRP1 (general receptor for 31980946 32171221phosphoinositides 1) 8. heterogeneous nuclear 38074994 51464712ribonucleoprotein K homolog 9. histidine triad nucleotide 3346885721359982 binding protein 10. immunoglobulin 9E10 2505942 (—) heavy chain11. KH-type splicing 38082738  4808586 regulatory protein 12. Profilin 112846944  4826898 13. prolyl 4-hydroxylase, 20913929 20070125 betapolypeptide 14. protein 40 kD homolog 38079048 51460993 15. proteinproduct 33115175 33115175 (—) 16. protein product 12845853 12845853 (—)17. RAD23a homolog 38089412 (—) 18. Ribosomal protein S12 17390846 (—)19. ring finger and WD 24418905 50233824 repeat domain 1 20.Serotransferrin precursor 21363012  136191 21. Synovial sarcoma, X21594537 41281571 breakpoint 2 interacting protein 22. Trk-fused gene19353070 21361320 23. Tumor rejection antigen gp96 15030324  4507677

All 20 of the spots differentially expressed in P16, i.e., present inP16 at greater than two-fold higher levels of expression relative to P1were excised and identified. From these 20 spots, 39 differentiallyexpressed proteins were identified (Table 2); four of them are unnamedprotein products and eight of them (Crybb1 protein, fatty acid bindingprotein 5, crystallin α1, crystallin βA1, crystallin βA4, crystallinβB3, crystalline γb, and crystalline γd) were identified in differentspots, as they represented modified versions or fragments of the sameprotein.

TABLE 2 Proteins Present in P16 at >2X levels of expression in P1 MOUSEHUMAN GENE HOMOLOG # PROTEIN BANK # GENE BANK # 1. protein product38089687 38089687 (—) 2. Acvr11 protein (ALK1) 15929282  4557243 3.Albumin 1 19353306  8392890 4. Alpha enolase 13637776  4433141 5.Cathepsin 3 precursor 19424144 (—) 6. Cofilin 1 37194891  5031635 7.Crybb1 protein 22137737 47678381 8. Crystallin, alpha A 30794510 (—) 9.Crystallin, beta A1 20304089 12056461 10. Crystallin, beta A2 3108896513623372 11. Crystallin, beta A4 33989574  4503059 12. Crystallin, betaB2 6681035  4503063 13. Crystallin, beta B3 37589234  4758074 14.Crystallin, gamma B 38049551  4885157 15. Crystallin, gamma C 3399060010518338 16. Crystallin, gamma D 34784220 13377002 17. Crystallin, gammaF 33991687 (—) 18. Cytochrome P450 2D9 homolog 38077646 (—) 19. Enolase3 15488630 16554592 20. Fatty acid binding protein 5 6754450 3058373621. FX3B_MOUSE F-box/ 37537783 (—) LRR-repeat protein 3B 22.Glyceraldehyde-3- 6679939 19924131 phosphate dehydrogenase 23. Guaninenucleotide-binding protein 15341782 20357526 24. Heat shock 70 kDprotein 5 29748016 24234686 25. Heat shock cognate 71 kDa protein 123651 123648 26. Hspb1 protein 17390597  4504517 27. IgM heavy chain 176374554780866 28. keratin 5b homolog 38077186 (—) 29. keratin complex 2,basic, gene 5 20911031 (—) 30. Knsl7 protein 38173736  9910266 31.protein product mKIAA0585 protein 28972291 (—) 32. protein productmKIAA0650 protein 37360026 (—) 33. protein kinase, cAMP 3154350938257138 dependent regulatory 34. protein product 1333921 1333921 (—)35. protein product 26335149 26335149 (—) 36. RAD50 homolog 667960919924130 37. Solute carrier family 20 28204938 58761541 38. Tubulin33416314 34784746 39. Vimentin 31982755 57471648II. Differential Protein Expression in P4 and P16 using IPG Strips pH4-7,5-8,7-10

The use of broad range strips allows the display of the most proteins ina single gel. With narrow-range and micro-range overlapping gradientstrips, resolution is increased by expanding a small pH range across theentire width of a gel and more spots can be displayed per each sample.This is due to the extra resolving power from use of a narrower pI rangeper gel. Because proteins outside of the pH range of the strip areexcluded, more total protein mass can be loaded per strip, allowing moreproteins to be detectable.

In this experiment, three samples for P4 and three samples for P16 wereloaded onto nonlinear immobilized pH gradient (IPG) 7 cm gel strips withpH 4-7, pH 5-8, pH 7-10 and rehydrated overnight with Rehydration/SampleBuffer. Isoelectric focusing (IEF) was performed with a programmedvoltage gradient at 8,000 V for 5 hours.

IPG strips were prepared for the second dimension by two sequential 10minute incubations in 6 M urea, 50 mM Tris (pH 8.8), 30% glycerol, 2%SDS, and 0.001% bromophenol blue, containing, alternately, 2% DTT or2.5% iodoacetamide.

Following equilibration, second-dimension separation was then performedon 4-20% SDS-PAGE gels (Biorad Laboratories, Hercules Calif.) with thefirst-dimension IPG strip embedded in 0.5% agarose at the top. Proteinson gel were fixed in 10% acetic acid and 20% methanol for 1 hour andwere then stained using SYPRO Rubin Protein Gel stain (BioradLaboratories, Hercules Calif.). In total, 12 gels with samples wereprepared and subjected to analysis for the first experiment and 6 forthe second experiment.

Using the Phoretix 2D imaging analysis software, the gels were warpedthe P4 map was matched with the P16 map to compare and identify proteinsthat were differentially expression in these gels. Of the proteins withdifferential expression, a number of spots were selected.

Comparing P4 map pH 4-7 with P16 map pH 4-7, fifteen spots were selectedthat included proteins that were differentially expressed found to beclearly present in P4 and barely detectable or not detectable in P16,and another fifteen spots that were clearly present in P16 and barelydetectable or not detectable in P1.

Comparing P4 map pH 5-8 with P16 map pH 5-8, three spots were found tobe clearly present in P4 and barely detectable or not detectable in P16,and another seven spots were clearly present in P16 and not in P4.

Comparing P4 map pH 7-10 with P16 map pH 7-10, two spots were found tobe clearly present in P4 and barely detectable or not detectable in P16,and another two spots were clearly present in P16 and barely detectableor not detectable in P4.

These 44 spots were identified using mass spectrometry analysis. Theresults are shown in Tables 3-4. Table 3 lists proteins that wereidentified as particularly interesting targets, due to their knownfunction.

TABLE 3 Potentially Relevant Proteins MOUSE HUMAN GENE HOMOLOG BANK GENEBANK # PROTEIN #GI #GI 1. protein BC031593 protein 17512384 (—) 2.protein Ppp3cc 12853166 13937367 3. 14-3-3 PROTEIN TAU homolog 12849349(—) (14-3-3 PROTEIN THETA) 4. 143B_MOUSE 14-3-3 protein 3065924 (—)beta/alpha 5. 143G_HUMAN 14-3-3 protein 3065928 (—) gamma 6. acidphosphatase 2, lysosomal 29150253  4557010 7. Acvr1 protein 15929282 4557243 8. annexin A5; annexin V 6753060  4502107 9. Ca<2+> dependentactivator 6753238 34452715 protein for secretion 10. calcium bindingprotein 2639022  7656952 11. Calnexin 56206506 10716563 12. cathepsin 3precursor 19424144 (—) 13. Cathepsin D 26101892  4503143 14. proteinAY100450; WFIKKN-like; 32451494 (—) growth and differentiation factor15. Cofilin 1 37194891  5031635 16. Creatine kinase, brain 10946574 (—)17. Cttn protein 15030315 21707902 18. development- and differentiation-51827426 (—) enhancing factor 2; PYK2 C homolog 19. DiGeorge syndromecritical region 29144908 (—) gene 6 product homolog 20. drebrin 1;drebrin E2; drebrin A 34328251 18426915 21. fatty acid binding protein5, 6754450  4503491 epidermal 22. Fbxo9 protein 18044861 53692184 23.fibroblast growth factor 22 12843402  120050 24. FK10_MOUSE FK506binding 26105984  7706131 protein 10 precursor 25. golgi associated,gamma adaptin 22122347 48527954 ear containing, ARF binding protein 126. GRP1 (general receptor for 26094386 32171221 phosphoinositides1)-associated scaffold protein 27. H2-T10 protein 18257363 (—) 28.Hepatoma-derived growth factor 34787412 55960780 29. Inner centromereprotein 26353663 51471706 30. integrin beta 2; integrin beta 2 26353439 4557886 (Cd18); Mac-1 beta; macrophage antigen 31. Male-specificlethal-3 homolog 1 11545735  5052315 32. Map3k4 protein 3759013957338496 33. Myristoylated alanine rich protein 6678768 57870608 kinaseC substrate 34. NRCA_MOUSE Neuronal cell 26333200 (—) adhesion moleculeprecursor (Nr-CAM) (NgCAM-related 35. PDZ domain containing homolog26325701 58533161 36. phosducin homolog 7684610  9943842 37. Profilin 112846944  4826898 38. protein 1200008A14 12835962 (—) 39. protein2310057D15Rik 12845067 (—) 40. protein 2610111M03Rik 12848227 (—) 41.protein 2700081O15Rik 26083118 (—) 42. protein A830054M12 26090077 (—)43. protein AI507495 47523976 (—) 44. protein AI663987 51708226 (—) 45.protein AU042671 18044599 (—) 46. protein BC037006 protein 17160955 (—)47. protein kinase, cAMP dependent 15030299  1346362 regulatory, type Ibeta 48. protein mKIAA0585 protein 28972291 (—) 49. protein mKIAA065012855134 (—) 50. protein mKIAA1565 26349512 (—) 51. protein NUF2R12963617 12667401 52. protein phosphatase 1, regulatory 12832826 (—)(inhibitor) subunit 2 homolog 53. Protein phosphatase 2, regulatory37718993  5453952 subunit B (B56), beta isoform 54. protein Prkcsh14602601 15488917 55. protein putative 40-2-3 protein 26346029 1521616256. protein Rbbp6 protein 30851332 49522749 57. protein tyrosinephosphatase, 25092609  2842713 receptor type, S 58.Ras-GTPase-activating protein 56800092 54695638 SH3-domain bindingprotein 59. SH3-domain GRB2 homolog 31560792 55661111 60. S-phasekinase-associated protein 1A 31560543 (—) 61. Tnf receptor-associatedfactor 3 6755865 22027620 62. Tnks protein 34980999 (—) 63. Tripartitemotif protein 28 59807693  3183179 64. Trk-fused gene 19353070 2136132065. Ubiquilin 2 34328236 17426453 66. ubiquitin-like 1 (sentrin)activating 7709986 (—) enzyme E1B; anthracycline-associated 67. Valosincontaining protein 30023842 55662798 68. zinc finger protein 120 isoform2 31044493 (—) 69. zinc finger protein 462; gene trap 26327260  8036504insertion site 4-2 70. Zyx protein 32451799 33870614 71. GRP1 (generalreceptor for 28277382 phosphoinositides 1)-associated scaffold 72.otoferlin [Mus musculus] 51831108 73. Cofilin 1, non-muscle 28374265[Mus musculus] 74. cathepsin 3 precursor 31560606 [Mus musculus] 75.Heat shock 70 kD protein 5 31981721 (glucose-regulated protein) [Musmusculus] 76. Acvrl1 protein [Mus musculus] 6752957 77. Tnfreceptor-associated factor 3 6755864 [Mus musculus] 78. integrin alpha2; integrin alpha 2 41054730 (Cd49b); VLA-2 receptor, alpha 2 79. H2-T10protein [Mus musculus] 51770266 80. fibrillin-1 precursor - mouse6755774 81. protein tyrosine phosphatase, 33286922 receptor type, S [Musmusculus] 82. transient receptor potential cation 59889895 channel,subfamily M, member 3 83. calcium channel, voltage- 51712446 dependent,L type, alpha 1S subunit 84. Krt1-15 protein 6680601 85. stress-inducedphosphoprotein 1; 14389430 stress-inducible protein; IEF SSP 352 86.Hepatoma-derived growth factor 31560690 87. Lmnb1 protein 6754555 88.Myristoylated alanine rich protein 6678767 kinase C substrate 89.phosphoglycerate kinase 30802044 90. acetyl-Coenzyme A 31542049acetyltransferase 1

Table 4 is a list of those proteins with unknown function that aredifferentially expressed during development of the HVS.

TABLE 4 Differentially Expressed Unknown Proteins MOUSE GENE SIGNIFICANTPossible function # PROTEIN BANK #GI HOMOLOGY with (by homology) 1.protein 4930477M19 29244200 solute carrier family22 2. protein XP_35837838089454 LOC348180 protein 3. protein XP_148779 38082098 unknown 4.protein 4732456N10 29244176 keratin 5 5. protein D630003M21 29244074KIAA1755 protein homolog 6. protein MGC11770 21450213 alanyl-tRNAsynthetase homolog 7. protein 4832416E03 27370264 alanyl-tRNA synthetasehomolog 8. protein S25715 284778 unknown protein - mouse 9. protein4932412H11 27370338 leucine-rich repeat containing protein homolog 10.protein product 26390223 protein disulfide 26390223 isomerase homolog11. protein product 26351281 Titin homolog 26351281 12. protein product1333921 crystallin, gamma A 1333921 13. protein product 26354026tropomyosin 3 26354026 14. protein product 26338367 unknown 26338367 15.protein product 26329025 unknown 26329025 16. protein product 12852979PDZ domain 12852979 containing 1 17. protein product 26325560 unknown26325560 18. protein product 26354901 ankyrin homolog 26354901 19.protein product 26340818 unknown 26340818 20. protein product 26346649tropomyosin 1 26346649 21. protein product 26346108 Interferon-induced26346108 protein 22. protein product 26335149 endothelial progenitor26335149 cells, angiopoietin homolog 23. protein product 38257031oogenesin 1 38257031 24. protein product 26342222 High mobility group26342222 homolog 25. protein product 26351025 unknown 26351025 26.protein for 38328278 heterogeneous nuclear MGC:70225 ribonucleoproteinA3 27. protein for 38328232 unknown MGC:69648 28. protein for 32451767Snx25 protein MGC:65613 29. protein for 38328220 septin 11 MGC:70304 30.protein for 38348540 unknown MGC:66590 31. protein for 38174349T-complex protein 10c MGC:74310 32. protein for 38173718 nestin homologMGC:66854 33. cDNA 4930513F16 29789229 IQ calmodulin-binding motifcontaining protein 34. cDNA 1110021E09 30354152 unknown 35. cDNA4930578C19 30424860 nucleotide apoptosis triphosphate hydrolases 36.cDNA 6030404K05 30425216 C2H2 zinc-finger at its gene N-terminal region37. cDNA C730025I08 30425272 RETINOL gene DEHYDROGENASE 38. cDNA2610019P18 30519939 unknown 39. cDNA C730027J19 30520215 rathypertension- gene associated homolog 40. cDNA 2610036L13 31127295 celldivision cycle angiogenesis associated 5 41. cDNA 5730444A13 31542010G-protein beta WD-40 apoptosis repeats 42. cDNA 5230400J09 33859785 dufcontaining peotein, zeta-crystallin 43. cDNA 1500011H22 34784371 unknown44. cDNA 9030623C06 34980904 keratin 21, type I, cytoskeletal 45. cDNA6430598A04 34980939 netrin-G1 ligand apoptosis gene NGL-1 46. cDNA1500031K13 37589970 calcium binding protein 39-like 47. cDNA 3110001E1138075065 unknown 48. cDNA 1700020H17 38075351 molybdopterin synthasesulfurylase 49. cDNA 4933432B09 38077919 unknown 50. cDNA 1110037F0238078070 unknown 51. cDNA 5730537H01 38084391 mRNA-decapping enzyme(DCP2) 52. cDNA E330005F07 38089151 microtubule binding gene proteinhook3, GTPase 53. cDNA B630009I04 38090459 activating signalcointegrator 1 complex 54. cDNA 9130006A14 38091346 Mus musculusproliferation RasGEF domain family, and apoptosis Cell Division ControlProtein 25 55. cDNA F830010122 38091563 TENSIN homolog 56. cDNAA930040G15 46048307 unknown 57. cDNA 4432411E13 51769098 hyperplasticdiscs protein, tumor suppressor 58. cDNA C130068M19 59858553 beta WD-40repeats containing protein

Differential Protein Expression in P1, P4, P8, P16 Using IPG Strips pH3-10 and Phosphostaining

In this experiment, two samples per each post-gestational day wereloaded onto nonlinear immobilized pH gradient (IPG) gel strips (7 cm, pH3-10) and rehydrated overnight with Rehydration/Sample Buffer (BioradLaboratories, Hercules, Calif.). The same steps were followed for thegel electrophoresis, and then proteins on gel were fixed in 10% aceticacid and 20% methanol overnight. Each of the 8 gels was incubated threetimes for 10 minutes with 100 mL of dH₂O with gentle agitation in orderto remove all of the methanol and acetic acid from the gel.

Then each gel was incubated in the dark in 50 mL of Pro-Q® Diamondphosphoprotein gel stain with gentle agitation for 75-120 minutes(Molecular Probes Laboratories); each gel was destained for two times in80-100 mL of Destain Solution (Molecular Probes Laboratories) at roomtemperature, protected from light. Images of each gel were taken withthe Image acquisition system Molecular Imager FX Pro Plus. Every gel wasthen stained for three hours with SYPRO Rubin Protein gel Stain (BioradLaboratories, Hercules Calif.), and proteins on the gel were fixed twicein 10% acetic acid and 20% methanol for 1 hour. Images of each gel weretaken with the Image acquisition system Molecular Imager FX Pro Plus.

The proprietary fluorescent stain of Pro-Q® Diamond phosphoprotein gelstaining (Molecular Probes Laboratories) allows direct in-gel detectionof phosphate groups attached to tyrosine, serine or threonine residues.The comparison between the images obtained from P1 and P16 gels stainedwith the phosphostain showed a different pattern of protein activation.Thirteen protein spot were identified with mass spectroscopy that werepresent in P1 but not in P16, and nine protein spots present in P16 butnot in P1. Proteins identified by this method are listed in Table 5.

TABLE 5 PROTEINS WITH ALTERATIONS IN PHOSPHORYLATION HUMAN MOUSE PROTEINGI # GI# 1810015P09Rik protein 21411262 2610111M03Rik protein 19353142 adisintegrin and metalloprotease domain 4; 27923590 a disintegrin andmetallop Albumin 1 19353306 alpha fetoprotein; alpha-foetoprotein31982513 antibody heavy chain variable region 1518293 B230317C12Rikprotein 37994630 Ca²⁺ dependent activator protein for 6753238 secretionCalnexin 25955477 CRAA_MOUSE Alpha crystallin A chain, 117369 majorcomponent Cryab protein 14789702 Crybb1 protein 22137737 crystallin,alpha A; lens opacity 18; 30794510 crystallin, alpha 1; alpha-A-crycrystallin, beta A1 20304089 Crystallin, beta A2 31088965 Crystallin,beta B3 37589234 crystallin, gamma B 38049551 Crystallin, gamma C33990600 Crystallin, gamma D 34784220 Crystallin, gamma F 33991687Crystallin, gamma S 46854370 or 33989585 47124529 drebrin 1; drebrin E2;drebrin A 34328251 dynein, axonemal, heavy polypeptide 9 38091406 Facl6protein 16359313 gamma 4-crystallin 51017 Heat shock 70 kD protein 5(glucose- 29748016 regulated protein) Heat shock protein 1, alpha28436908 heat shock protein 1, beta; heat shock 6680305 protein, 84 kDa1; heat shock 90 heat shock protein, 110 kDa 13278232 Hook homolog 138197327 HS7C_MOUSE Heat shock cognate 71 123651 kDa protein Hspcbprotein 29612561 hypothetical protein 4732456N10 29244176 IgM heavychain 1763745 integrin alpha 2; integrin alpha 2 (Cd49b); 6680478 VLA-2receptor, alpha 2 sub laminin, beta 2; Laminin S 31982223 laminin, gamma2; nicein, 100 kD; nicein, 19115956 100 kDa Lmnb1 protein 34849832mannosidase 2, alpha 2; alpha 51477716 27777691 mannosidase IIx;mannosidase, alpha, cla mKIAA0723 protein 37360062 mutS homolog 57305281 Myristoylated alanine rich protein 28302374 kinase C substrateNasp protein 13435642 nuclear autoantigenic sperm protein 13384598Pleckstrin homology-like domain, family 17512344 A, member 2prolactin-like protein E; prolactin-like 6679471 protein G proteintyrosine phosphatase, receptor 25092609 type, S PSD3_MOUSE 26Sproteasome non-ATPase 19856169 regulatory subunit 3 (26S proteasome regRbbp6 protein 30851332 Ribosomal protein S27a 12805285 RIKEN cDNA0610041L09 21313618 RIKEN cDNA 1500011H22 34784371 RIKEN cDNA 1700028P1429436989 RIKEN cDNA 3000003F02 27369583 RIKEN cDNA 4432411E13 38077035RIKEN cDNA 6030404K05 gene 30425216 RIKEN cDNA A030005L19 13386450 RIKENcDNA D430026P16 gene 28893017 RIKEN cDNA F830010I22 gene 38091563 S25715hypothetical protein - mouse 284778 (fragment) Serine (or cysteine)proteinase inhibitor, 19343549 clade A, member 1b [Mus muscu Serine (orcysteine) proteinase inhibitor, 38174657 clade A, member 1e [Mus muscuSerpina1b protein 15277553 similar to 60S ribosomal protein L10 (QM38079574 protein homolog) [Mus musculu similar toalpha-2-HS-glycoprotein 38080571 homolog - mouse similar toalpha-tubulin 38050521 similar to ATP-binding cassette, sub-family38049559 A, member 12 isoform b [Mu similar to Crybb1 protein 38074960similar to Elongation factor 1-alpha 1 38090266 (EF-1-alpha-1)(Elongation fact similar to heat shock protein hsp90 beta 38096689similar to hypothetical protein FLJ21665 38074149 similar to K-ALPHA-1protein 38084290 Similar to neuronal protein 28175466 similar to tubulinMbeta 1 38075765 similar to tubulin, beta 3 38085389 Solute carrierfamily 3, member 1 18490867 15488595 S-phase kinase-associated protein1A 12805297 Synovial sarcoma, X breakpoint 2 21594537 interactingprotein Tnks protein 34980999 Tripartite motif protein 28 37231553trypsinogen 10 2358087 trypsinogen 16 16716569 Tubulin, alpha 2 12805487Tubulin, alpha 6 20071240 tubulin, beta 21746161 Tubulin, beta 233416314 Tubulin, beta 3 21595026 Tubulin, beta 4 32766247 Tubulin, beta5 13277909 ubiquitin specific protease 9, Y 22507351 chromosomeubiquitin-like 1 (sentrin) activating 7709986 enzyme E1B;anthracycline-associa Unknown (protein for MGC:69991) 38328337 unnamedprotein product 1333921 Valosin containing protein 29144989 vimentin57471648 31982755 ZIN_MOUSE Striatin 4 (Zinedin) 17367523 1012270Bantibody L-V, anti-arsonate GI:224215 2700081O15Rik protein 375895815730509E04Rik protein 20071529 Actin-like 6 29436558 arylalkylamineN-acetyltransferase 6752938 ATPase, H+ transporting, V1 subunit A,31560731 isoform 1; ATPase, H+ transport beaded filament structuralprotein 2, 38090061 phakinin Cathepsin D 21450788 CRAA_MOUSE Alphacrystallin A chain, 117369 major component Creatine kinase, brain15929689 Cryab protein 14789702 Crybb1 protein 22137737 crystallin,alpha A; lens opacity 18; 30794510 crystallin, alpha 1; alpha-A-crycrystallin, beta A1 20304089 Crystallin, beta A2 31088965 Crystallin,beta A4 33989574 crystallin, beta B2; betaB2-crystallin; 6681035 Phillycataract Crystallin, beta B3 37589234 crystallin, gamma B 38049551Crystallin, gamma C 33990600 Crystallin, gamma D 34784220 Crystallin,gamma F 33991687 crystallin, gamma N 23346485 Crystallin, gamma S33989585 eukaryotic translation initiation factor 3, 21313620 subunit 5(epsilon) [Mus m G protein-coupled receptor 106; G protein 15546059coupled receptor affecting t gamma 4-crystallin 51017 Hnrpk protein13879427 HS7C_MOUSE Heat shock cognate 71 kDa 123651 proteinhypothetical protein 4932412H11 26325921 integrin alpha 2; integrinalpha 2 (Cd49b); 6680478 VLA-2 receptor, alpha 2 sub kinaseD-interacting substance of 220 kDa 38049418 Kinesin-like 6 13905108laminin, beta 2; Laminin S 31982223 Map3k4 protein 37590139 mKIAA1565protein 37360452 Nitrogen fixation cluster-like 29476869 olfactoryreceptor GA_x6K02T2PBJ9- 32057602 6773690-6774628 phakinin, CP4917977856 PSD3_MOUSE 26S proteasome non-ATPase 19856169 regulatorysubunit 3 (26S proteasome reg RIKEN cDNA 4833420I20 17390408 RIKEN cDNA4930578C19 30424860 RIKEN cDNA 4933424A10 gene 28893379 RIKEN cDNA6030404K05 gene 30425216 RIKEN cDNA 9030623C06 34980904 RIKEN cDNAA030005L19 13386450 RIKEN cDNA A930040G15 19527136 RIKEN cDNA C130068M19gene 38073604 RIKEN cDNA C730027J19 gene 30520215 RIKEN cDNA F830010I22gene 38091563 Sema domain, immunoglobulin domain 37046950 (Ig), and GPImembrane anchor, (semap SH3-domain GRB2-like 2 17390906 similar to14-3-3 protein sigma 38089318 similar to actin, gamma, cytoplasmic38089775 similar to cytoplasmic beta-actin 38089227 similar to fibroussheath-interacting 38074908 protein 2 similar to integrin alpha 638090030 similar to ribosomal protein L31 38085230 solute carrier family6 (neurotransmitter 19527208 transporter, creatine), memb T-box 120891977 trypsinogen 16 16716569 Tubulin, alpha 2 12805487 uncouplingprotein 2, mitochondrial 31543920 Unknown (protein for MGC:66590)38348540 Unknown (protein for MGC:69991) 38328337 Unknown (protein forMGC:74310) 38174349 unnamed protein product 26335149 unnamed proteinproduct 1333921 unnamed protein product 26351281 unnamed protein product1333921

Example 2 Differential Expression of Activin Receptor-Like Kinase 1(ALK1)

The activin receptor-like kinase 1 (ALK1), a TGF-beta1 type I receptor,plays an inhibitory role in angiogenesis and vascular development.Mutations of ALK1 gene are linked to human type II hereditaryhemorrhagic telangiectasia. Our purpose was to develop a mouse model tostudy the differential expression of proteins during the phase ofHyaloid Vascular System (HVS) regression and determine the role of ALK1in this model.

Materials and Methods: Thirty-two newborn C57BL/6 mice were sacrificedon post-gestational days 1 (n=20 eyes), 4 (n=20 eyes), 8 (n=12 eyes),and 16 (n=12 eyes). The lens, the Pupillary Membrane (PM), the TunicaVasculosa Lentis (TVL) and the primary vitreous containing the VasaHyaloidea Propria (VHP) were isolated. Proteins were extracted from eachspecimen, loaded onto nonlinear immobilized pH gradient (IPG) gelstrips, and separated by isoelectric points and molecular weights.Protein expression profile at each time point was compared using thePhoretix 2D image analysis software. Proteins from differentiallyexpressed protein spots were isolated and identified using MassSpectrometry (MS). Immunohistochemistry was performed to determine theexpression of ALK1 during the HVS regression phase.

Results: The generated protein expression maps showed reproducibleseparation of the protein spots on the 2 Dimension Electrophoresis gels.Up to 1400 proteins spots were detected per gel. Progressive decrease inthe number and intensity of the protein spots occurred from P1 to P16,particularly in the area corresponding to pI 4-7 and M_(r) 30 −90 kDa.Twenty protein spots in the P16 gels were not present in the P1 gels.

MS revealed 39 differentially expressed proteins (PP 16-1 to 39) in theP16 specimens (Table 1). ALK1 was identified as PP 16-31 (spot n° 17).The warping of P16 with P1, P4 and P8 showed the presence of this spotonly in P4 and P8. Immunohistochemistry of the cornea, PM, and TVL usinganti ALK1 antibody confirmed the presence of ALK1 in the TVL at P4 andP16.

Conclusion: The synthesis or degradation of one or more proteins,present at P16 (PP16-1 to 39), may be related to the regression of HVSin the mouse. Identification of ALK1 by proteomic analysis andimmunohistochemistry in this model suggests that the TGF-beta1 pathwaymay be involved in this process.

Example 3 Progressive Regression of HVS in the Mouse and theDifferential Protein Expression Profile

A time course experiment was designed to examine the changes in proteinexpression at P1 and P16 throughout the HVS regression process. As thisis the first reported application of proteomic analysis on HVSregression, several conditions for tissue selection and preparation weredeveloped and optimized to obtain clean specimens of the lens andvitreous for 2-DE gels.

Mouse Anterior Tissue Preparation for 2-DE Gels

For the protein extraction and ALK1 immunolocalization studies, newbornmice were sacrificed on post-natal days 1, 4 and 16. Using a dry icebed, frozen mouse eyes were scraped with a blade (Beaver #15) to removethe cornea, conjunctiva, sclera, ciliary body, uvea, and retina, leavingthe lens surrounded by the PM, TVL, and primary vitreous (containingVHP). The dry ice bed was used to prevent warming of the specimen,tissue melting, and subsequent protein denaturation. Samples of P1, P4and P16 were pooled and independently solubilized in 250 μl of totalprotein extraction buffer 7 M urea, 2 M thiourea, 1% (w/v) ASB-14detergent, 40 mM Tris base, 0.001% bromophenol blue, 20% carrierampholyte (Biorad Laboratories, Hercules, Calif.) and 2 μl of 200 mMtributylphosphine by mechanically homogenizing them with an electricaltissue homogenizer for 5 min on ice. The homogenates of the proteinextracts were cleared by centrifugation for 20 min at 14,000 rpm and 4°C. to remove particulates. Protein concentration of the supernatants wasdetermined by protein assay (Biorad Laboratories).

Two-Dimensional Electrophoresis, Image Analysis and ProteinIdentification

Three samples per each post-gestational day were loaded onto nonlinear,immobilized pH gradient (IPG) gel strips (7 cm, pH 3-10), subjected toisoelectric focusing with a programmed voltage gradient at 8,000 V for 5hours, and rehydrated overnight with rehydration/sample buffer (BioradLaboratories). IPG strips were equilibrated with buffer containing 6 Murea, 50 mM Tris (pH 8.8), 30% glycerol, 2% SDS (sodium dodecylsulfate), and 0.001% bromophenol blue containing 2% DTT (dithiothreitol)or 2.5% iodoacetamide. Following equilibration, second-dimensionseparation was performed on 4-20% SDS-PAGE (SDS-polyacrylamide gelelectrophoresis) gels (Biorad Laboratories) with the first-dimension IPGstrip embedded in 0.5% agarose at the top. After electrophoresis, theproteins on the gel were fixed in 10% acetic acid and 20% methanol for 1hour and then stained with SYPRO™ Ruby (Biorad Laboratories). Images ofthe 2-DE (two-dimensional electrophoresis) gels were captured withMolecular Imager FX Pro Plus multi-imager system and the proteinexpression profiles at each time point were compared, in triplicate,using the Phoretix 2D image analysis software. Protein spots present inP16 with expression level two- to ten-fold greater than P1 wereconsidered for protein identification. The statistical significance ofchanges was evaluated using Phoretix 2D software.

Gel bands (differentially expressed proteins) from 2-DE gels wereexcised, minced into approximate 1 mm³ pieces with a sterile razor bladeof the Xcise technology platform from Proteome system, alkylated, anddigested with trypsin (Promega, Madison, Wis.). Peptides wereresuspended in 50% acetonitrile with 1% formic acid solution andsubjected to nano-LC ESI IT MS/MS analysis. A Surveyor LC pump (ThermoElectron, San Jose, Calif.) with a C18 trapping column (300 μm i.d.×1mm, Dionex, Sunnyvale, Calif.) and a reversed-phase column 75 μm i.d.×15cm (Magic C18AQ, 3 μm) was used for nano-LC experiments. An LCQ Deca XPplus ESI mass spectrometer (Thermo Electron, San Jose, Calif.) was usedfor all the experiments.

MS/MS data-dependent acquisition followed by database searching withSEQUEST (BioWorks 3.1, Thermo Electron, San Jose, Calif.) allowedprotein identification. Fully tryptic peptides were matched with SEQUESTat a delta correlation (ÄCn) of greater than 0.08 and correlation(Xcorr) greater than 1.9, 2.2, and 3.5 for charged states of +1, +2, and+3, respectively. Peptide mass fingerprints (PMFs) were searched formatches with the virtually generated tryptic protein masses of theNCBInr mouse protein sequence database (available at ncbi.nml.nih.gov).All databases were provided in the public domain by the hostinstitutions. Proteins were noted as differentially expressed if theycould not be located at the corresponding position on the 2-DE gel ofthe other time point.

Protein expression maps were generated in triplicate for each timepoint, obtaining a reproducible separation of the protein spots on the2-DE gels. Up to 1400 protein spots could be detected in each gel withthe Phoretix 2D image analysis software (FIG. 1A). This software allowsthe possibility to warp different gel images to improve the quality ofthe protein spot matching between the gels and allow an easy detectionof the differences among the gels. The 2-DE gels were analyzed, and aprogressive decrease in the number and intensity of the protein spotsfrom P1 to P16 was observed, particularly of the migrated proteins inthe area corresponding to pI 4-7 and M_(w) 30-90 kDa (FIG. 1B-C).

However, the warping of the 2-D gel images of P1 with P16 revealed thepresence of a small number of protein spots for which expression levelin P16 was two to ten-fold greater than P1. Twenty protein spots wereanalyzed using mass spectrometry analysis. Some of these identifiedproteins displayed slightly different molecular weights and pIs,suggesting the presence of different isoforms or posttranslationalmodifications. Eighteen differentially expressed proteins wereidentified; two additional proteins were unclassified and 15 wereunknown. ALK1 was identified as spot #17.

TABLE 6 PROTEINS WITH DIFFERENTIAL EXPRESSION AT POST-GESTATIONAL DAY 16MOUSE HUMAN GENE HOMOLOG SPOT # PROTEIN NAME BANK # GENE BANK # 1 RAD50homolog 6679609 19924130 2 Olfactory receptor 32057602 41200972GA_x6K02T2PBJ9-6773690- 6774628 3 Cofilin 1, non-muscle 28374265 1168484 Guanine nucleotide-binding 15341782 20357526 protein, β-1 subunit 5Protein kinase, cAMP dependent 15030299 1346362 regulatory, type I β 6 αenolase (2-phospho-D- 13637776 119339 glycerate hydro-lyase) (non-neural enolase) 7 Cathepsin 3 precursor 21450788 30582658 8 Unclassified9 Knsl7 protein 26095143 Knsl7 10 F-box/LRR-repeat protein 3B 3753778316306584 (F-box and leucine-rich repeat protein) 11 Enolase 3, β muscle15488630 16554592 12 Albumin 1 19353306 113576 13 Heat shock cognate 71kDa 123651 123648 protein 14 Fatty acid binding protein 5, 67544504557581 epidermal; keratinocyte lipid binding protein 15 Vimentin31982755 57471648 16 Tubulin, β 2 33416314 4507729 17 ALK1 protein6752957 4557243 18 CRAA_MOUSE α crystallin 117369 1706112 A chain, majorcomponent 19 Crystallin, β A4 33989574 4503059 20 Unclassified

Example 4 Immunolocalization of ALK1 in the TVL by Confocal Microscopy

To investigate whether ALK1 is expressed in TVL during mousedevelopment, mouse eyes at P1, P4 and P16 were immunostained withanti-ALK1 antibody.

ALK1 Immunolocalization by Confocal Microscopy

Mouse eyes harvested at P1, P4, and P16 were embedded in optimal cuttingtemperature (OCT) compound (Miles, Elkhart, Ind.), frozen in liquidnitrogen, cryostat sectioned, and fixed in acetone for 10 min. Afterblocking with 1% bovine serum albumin (BSA) (Sigma Chemical Co., St.Louis, Mo.) for 30 min, the sections were incubated for 1 hour withrabbit anti-ALK1 polyclonal antibody (courtesy of Dr. D. A. Marchuck,University of North Carolina) at a concentration of 1:200. A secondaryantibody used was fluorescein-conjugated, affinity-purified anti-rabbitIgG antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.) at aconcentration of 1:400. Negative controls were prepared in the samemanner, with 1% BSA without primary antibody. Immunostained sectionswere viewed with a Leica TCS SP2 CLSM confocal laser scanning microscope(Leica, Heidelberg, Germany).

Results

The confocal immunohistochemical staining demonstrated that ALK1 islocalized in the endothelial cells of the TVL at P1, P4 and P16.

Example 5 Naked pEF-ALK1 DNA Blocked bFGF Induced Corneal Nev. in Vivo

Naked DNA has been effectively used for the delivery of DNA into mousecorneas. To determine whether ALK1 can inhibit bFGF-induced corneal NV(neovascularization), mouse corneas were injected with naked DNAs of pEF(vector) and pEF-ALK1.

Plasmid Construction and Corneal Naked DNA Injection

pYX-Asc-ALK1 cDNA was purchased from ATCC (Manassas, Va.). Plasmids werepurified using a Qiagen kit (Valencia, Calif.) according to themanufacturer's instructions. The mouse ALK1 DNA was sequenced andsubcloned into pEF expression vector. An Elongation Factor 1 alphapromoter (pEF; Kim et al., Gene. 1990 Jul. 16; 91(2):217-23) was used todrive the expression of the ALK1 gene. Mouse corneas received a DNAinjection of 5 μl of pEF-ALK1 (400 ng/μl) or an injection of 5 μl ofvector (400 ng/μl). An antibiotic ophthalmic ointment was used aftersurgery.

bfGF Pellet Preparation and Corneal Micropocket Assay

Pellets were made of the slow-release polymer Hydron(polyhydroxyethyl-methacrylate), which contained a combination of 45ng/pellet of sucralfate (Sigma) and 120 ng/pellet of bFGF (R&D Systems,Minneapolis, Minn.), as previously described by Kenyon et al., InvestOphthalmol V is Sci 37, 1625-1632 (1996).

Briefly, a suspension of sterile saline containing the appropriateamount of recombinant bFGF and sucralfate was made and concentratedusing a speed-vacuum for 8 minutes. To this suspension, 10 μl of 12%Hydron in ethanol was added. The suspension was then deposited onto asterilized nylon mesh (LAB Pak, Sefar America, Depew, N.Y.) and embeddedbetween the fibers. The resulting grid of 10 mm×10 mm squares wasallowed to dry on a sterile Petri dish for 60 min. The fibers of themesh were pulled apart under a microscope and, among the approximately100 pellets produced, 30 to 40 uniformly-sized pellets of 0.4×0.4×0.2 mmwere selected for implantation. All procedures were performed understerile conditions.

Three days after the naked DNA injections, corneal micropockets werecreated with a modified von Graefe knife in C57BL/6 mice. Hydron pelletscontaining 120 ng of human basic FGF were implanted into the cornealpockets.

Measurement of Corneal Neovascularization

The corneas were routinely examined and photographed in five positions:en face, superior, inferior, nasal, and temporal with a slit lampbiomicroscope (Nikon FS-2, Tokyo, Japan) on days 1, 4, 7, and 14post-pellet implantation. The photographs were digitized and the imageswere resolved at 300 pixels/inch and analyzed with the NIH ImageJ imageprogram (NIH, Bethesda, Md.). NIH ImageJ program was used to calculatethe area of corneal neovascularization areas. Statistical analysis wasperformed with the student T-test.

Results

The ALK1 plus pellet group (FIG. 2M-P) showed no evidence of corneal NVat any time point after pellet implantation. The group receiving vectorcontrol plus pellets

(FIG. 2I-L) began to develop corneal NV at day 4, and the new vesselscontinued to grow toward the pellet area at days 7 and 14. The areaoccupied by neovascularization in the ALK1 treated mice was 0.58 mm² and0.47 mm² at days 7 and 14 after bFGF pellet implantation, respectively(FIG. 2O-P). This was significantly less than the vector treated mice3.14 mm² (p=0.001) and 3.33 mm² (p=0.001) at 7 and 14 days after bFGFpellet implantation, respectively (FIG. 2K-L). The no-pellet controlgroups [ALK1 naked DNA (FIG. 2E-H) and vector naked DNA (FIG. 2A-D)] didnot induce corneal NV.

These results demonstrate that injection of naked pEF-ALK1 DNA blockedbFGF induced corneal NV.

ADDITIONAL REFERENCES

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Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for identifying a candidate compound for the treatment of adisorder associated with neovascularization, the method comprising:providing a sample comprising a cell expressing activin receptor-likekinase-1 (ALK1); contacting the sample with a test compound; andmonitoring expression of ALK1 in the cell, wherein a test compound thatincreases the expression of ALK1 is a candidate compound for thetreatment of neovascularization.
 2. A method for identifying a candidatetherapeutic agent for the treatment of a disorder associated withneovascularization, the method comprising: providing an animal model ofthe disorder; administering a candidate compound that increases theexpression of activin receptor-like kinase-1 (ALK1) to the animal model;and monitoring one or more parameters of the disorder in the animal,wherein a candidate compound that improves a parameter of the disorderis a candidate therapeutic agent for the treatment of a disorderassociated with neovascularization.
 3. A method of identifying atherapeutic agent for the treatment of a disorder associated withneovascularization, the method comprising: providing a subject havingthe disorder; administering a candidate therapeutic agent that increasesthe expression of activin receptor-like kinase-1 (ALK1) to the subject;and monitoring one or more parameters of the disorder in the subject,wherein a candidate therapeutic agent that improves a parameter of thedisorder is a therapeutic agent for the treatment of a disorderassociated with neovascularization.
 4. The method of claim 1, whereinthe sample comprises cells from a human fetus.
 5. The method of claim 1,wherein the sample comprises cells from a subject having a disorderassociated with blood vessel regression.
 6. The method of claim 5,wherein the disorder is retinopathy of prematurity or retrolentalfibroplasia.
 7. The method of claim 1, wherein the sample comprisescells from a subject in a stage of development that is associated withvessel regression.
 8. The method of claim 7, wherein the stage is thedevelopment of the hyaloid vascular system.
 9. The method of claim 3,wherein the subject is a human subject in a clinical trial.
 10. Themethod of claim 1, wherein the disorder associated withneovascularization is selected from the group consisting of extraocularand ocular neovascularization.
 11. The method of claim 10, wherein theocular neovascularization is vitreous, retinal, choroidal, or cornealneovascularization.
 12. The method of claim 1, wherein the disorderassociated with neovascularization is cancer.
 13. A method for treatinga disorder associated with neovascularization in a subject, the methodcomprising administering a therapeutically effective amount of atherapeutic composition comprising an activin receptor-like kinase-1(ALK1) polynucleotide or polypeptide, or an active fragment thereof. 14.The method of claim 13, wherein the composition comprises an ALK1polypeptide comprising amino acids 22-503 of SEQ ID NO:1, or an ALK1nucleic acid molecule comprising nucleotides 346-1791 of SEQ ID NO:2.15. The method of claim 13, wherein the disorder is an ophthalmologicaldisorder associated with neovascularization.
 16. The method of claim 15,wherein the ophthalmological disorder associated with neovascularizationis selected from the group consisting of eye cancer, age-related maculardegeneration, retinopathy of prematurity, corneal graft rejection,glaucoma, diabetic retinopathy, wounds, age-related maculardegeneration, herpetic and infectious keratitis, ocular ischemia,neovascular glaucoma, corneal, uveal and iris neovascularization,orbital and eyelid tumors, Stevens Johnson Syndrome, ocular cicatricialpemphigoid, and ocular surface diseases.
 17. The method of claim 15,wherein the ophthalmological disorder is associated with corneal,retinal, choroidal, uveal, or iris neovascularization.
 18. The method ofclaim 15, wherein the administering is topical or parenteraladministration into the eye.
 19. The method of claim 17, wherein theadministering is by local injection into or near the cornea, retina,vitreous, uvea, orbit, eyelid, conjunctiva, or iris.
 20. The method ofclaim 13, wherein the disorder is cancer.